Regulation of eukaryotic gene expression involves the precise control of chromatin structure. While the manipulation of the chromatin fiber to create and maintain distinct functional domains is poorly understood, histone post-translational modifications are clearly central to this process. The long-range objective of this proposal is to elucidate roles of histone methylation in chromatin function. To achieve this, we will characterize the functions of two independent histone methyltransferase (HMT) activities recently identified in yeast. One of these activities is Set2, which is the sole enzyme in yeast responsible for methylating histone H3 on lysine 36 (K36), a site of modification conserved from yeast to humans. New evidence suggests that Set2-mediated H3 K36 methylation participates in glucose repression, but little else is known about this enzyme or its methylation site. Given recent insights into the roles of histone methylation, we hypothesize that these enzymes regulate gene expression, in part, through the recruitment of down-stream regulatory factors to their cognate methylation sites in chromatin. To that end, we will use a combination of biochemistry, genetics and immunology to define the domains in Set2 responsible for gene repression and histone methylation, characterize Set2's interacting partners, identify proteins that bind K36-methylated H3 and determine the extent to which H3 K36 methylation functions with other histone modifications to regulate the transcription of a gene affected by Set2. We will also determine other chromosomal regions and cellular pathways affected by Set2/H3 K36 methylation in an effort to understand the global functions of this enzyme. The second HMT activity is not yet identified but has been highly purified by chromatography. We will identify this enzyme by biochemical approaches and further characterize its site(s) of methylation and determine the extent to which they occur in chromatin. Since histone modifications play a vital role in chromatin-based functions, advances in our understanding of the enzymes that mediate them, as well as their functional significance, is likely to have a major impact on issues related to human health and disease.